Elevation of free fatty acids (FFA) results in an increase in the intracellular Ca2+ concentration of the a-cell. This effect does not appear to involve alterations in membrane potential, but is consistent with the augmented insulin release observed with acute exposure to FFA. This rise in intracellular Ca2+ was inhibited by dihydropyridines and thus depended on functional L-type (voltage-dependent calcium channels) VDCC. We propose that FFA elicit an acute response of the VDCC, increasing the intracellular Ca2+ concentration and ultimately insulin secretion. Our preliminary data from the fluorescence resonance energy transfer analyses indicate that the L-type channel predominantly expressed in beta-cells, the neuroendocrine alpha1D subunit, preferentially associates with a beta2a isoform found to be palmitoylated in other cell systems. Thus, we hypothesize that the FFA-mediated response may be exerted by a direct modulatory effect on the L-type alpha1D-beta2a channel through acylation of the beta2a subunit. We propose to discern the molecular mechanisms underlying the acute FFA-induced, L-type channel-dependent increase in intracellular Ca2+, as well as the cellular response(s) following long term exposure to FFA, using a combination of cellular, molecular and electrophysiological approaches. The specific aims are: 1. To determine the cellular mechanisms underlying the FFA-induced Ca2+ rise in a-cells. We will quantitate the FFA-induced changes in Ca2+ handling in beta-cell lines by assessing changes in unidirectional Ca2+ fluxes and calcium content. At the single cell level we will evaluate the heterogeneity, chain length specificity and concentration dependence of the Ca2+ response induced by FFA. We will establish whether these effects are exerted by FFA or its activated form, long chain acyl-CoA and distinguish between direct binding and protein acylation. 2. To determine the VDCC molecular components responsible for the FFA-induced effects. We will identify in COS-7 cells expressing various combinations of fluorescent-labeled alpha1 and beta subunit isoforms the L-type VDCC subunit(s) that, as the target of FFA action, is responsible for the DHP-inhibitable Ca2+ rise. We will generate chimeric and site-directed mutants of the subunit(s) to pinpoint the residues involved in the interaction. We will examine the effects of the mutations on FFA-mediated augmentation of insulin secretion in INS-1 cells. 3. To determine the effects of FFA on the electrical activity of VDCC. We will perform electrophysiological assays in beta-cell lines to measure the direct effects of FFA on currents through L-type channels and to determine the mechanism by which FFA enhance inward currents through those VDCC. We will separate the effects of FFA on different L-type VDCC and determine the effects of FFA on currents through mutant channels generated in Aim 2.